Function of Cajal Bodies in Nuclear RNA Retention in A. thaliana Leaves Subjected to Hypoxia.

Retention of RNA in the nucleus precisely regulates the time and rate of translation and controls transcriptional bursts that can generate profound variability in mRNA levels among identical cells in tissues. In this study, we investigated the function of Cajal bodies (CBs) in RNA retention in A. thaliana leaf nuclei during hypoxia stress was investigated. It was observed that in ncb-1 mutants with a complete absence of CBs, the accumulation of poly(A+) RNA in the leaf nuclei was lower than that in wt under stress. Moreover, unlike in root cells, CBs store less RNA, and RNA retention in the nuclei is much less intense. Our results reveal that the function of CBs in the accumulation of RNA in nuclei under stress depends on the plant organ. Additionally, in ncb-1, retention of introns of mRNA RPB1 (largest subunit of RNA polymerase II) mRNA was observed. However, this isoform is highly accumulated in the nucleus. It thus follows that intron retention in transcripts is more important than CBs for the accumulation of RNA in nuclei. Accumulated mRNAs with introns in the nucleus could escape transcript degradation by NMD (nonsense-mediated mRNA decay). From non-fully spliced mRNAs in ncb-1 nuclei, whose levels increase during hypoxia, introns are removed during reoxygenation. Then, the mRNA is transferred to the cytoplasm, and the RPB1 protein is translated. Despite the accumulation of isoforms in nuclei with retention of introns in reoxygenation, ncb-1 coped much worse with long hypoxia, and manifested faster yellowing and shrinkage of leaves.

EPIP as an abscission promoting agent in the phytohormonal pathway.

Understanding the mechanisms underlying the activation of the abscission zone (AZ) responsible for organ separation from plant body in crop species will help improve their yielding and economic importance. Special attention has been given recently to the role of the INFLORESCENCE DEFICIENT IN ABSCISSION protein, particularly its functional fragment, EPIP peptide. Its stimulatory effect on abscission in different crops has been demonstrated. Recently we described the role of EPIP in the redox, lipid, and pectin-related events taking place in AZ of Lupinus luteus flowers, which undergo massive abscission in natural conditions. To further examine EPIP contribution in AZ functioning, here, we analyze its impact on the ultrastructural changes, synthesis of two hormonal abscission stimulators - abscisic acid (ABA) and ethylene (ET), and the appearance of phosphoproteins. As our results show, the response of flower AZ to exogenous EPIP involves the induction of distinct modifications related to the one hand with upregulation of cell activity but on the other hand degradation processes and possible autophagy. Furthermore, the EPIP stimulated biosynthesis pathways of ABA and ET precisely in AZ cells. In addition, progressive phosphorylation of proteins has been observed under EPIP influence. The highly accumulated ones were identified as those, related to primary metabolism and reactive oxygen species homeostasis, and their role in abscission has been discussed. To summarizing, the presented detailed description of EPIP action in AZ cells in combination with our previous data offers new insights into its regulatory function and provides opportunities to counteract excessive flower abscission in lupine.

MicroRNA biogenesis and activity in plant cell dedifferentiation stimulated by cell wall removal.

BACKGROUND: Despite the frequent use of protoplast-to-plant system in in vitro cultures of plants, the molecular mechanisms regulating the first and most limiting stages of this process, i.e., protoplast dedifferentiation and the first divisions leading to the formation of a microcallus, have not been elucidated. RESULTS: In this study, we investigated the function of miRNAs in the dedifferentiation of A. thaliana mesophyll cells in a process stimulated by the enzymatic removal of the cell wall. Leaf cells, protoplasts and CDPs (cells derived from protoplasts) cultured for 24, 72 and 120 h (first cell division). In protoplasts, a strong decrease in the amount of AGO1 in both the nucleus and the cytoplasm, as well as dicing bodies (DBs), which are considered to be sites of miRNA biogenesis, was shown. However during CDPs division, the amounts of AGO1 and DBs strongly increased. MicroRNA transcriptome studies demonstrated that lower amount of differentially expressed miRNAs are present in protoplasts than in CDPs cultured for 120 h. Then analysis of differentially expressed miRNAs, selected pri-miRNA and mRNA targets were performed. CONCLUSION: This result indicates that miRNA function is not a major regulation of gene expression in the initial but in later steps of dedifferentiation during CDPs divisions. miRNAs participate in organogenesis, oxidative stress, nutrient deficiencies and cell cycle regulation in protoplasts and CDPs. The important role played by miRNAs in the process of dedifferentiation of mesophyll cells was confirmed by the increased mortality and reduced cell division of CDPs derived from mutants with defective miRNA biogenesis and miR319b expression.

Subcellular Localization of Acyl-CoA: Lysophosphatidylethanolamine Acyltransferases (LPEATs) and the Effects of Knocking-Out and Overexpression of Their Genes on Autophagy Markers Level and Life Span of A. thaliana.

Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence.

Spatial and Temporal Distribution of Arabinogalactan Proteins during Larix decidua Mill. Male Gametophyte and Ovule Interaction.

The role of ArabinoGalactan Proteins (AGPs) in the sexual reproduction of gymnosperms is not as well documented as that of angiosperms. In earlier studies, we demonstrated that AGPs play important roles during ovule differentiation in Larix decidua Mill. The presented results encouraged us to carry out further studies focused on the functions of these unique glycoproteins during pollen/pollen tube and ovule interactions in Larix. We identified and analyzed the localization of AGPs epitopes by JIM4, JIM8, JIM13 and LM2 antibodies (Abs) in male gametophytes and ovule tissue during pollination, the progamic phase, and after fertilization and in vitro growing pollen tubes. Our results indicated that (1) AGPs recognized by JIM4 Abs play an essential role in the interaction of male gametophytes and ovules because their appearance in ovule cells is induced by physical contact between reproductive partners; (2) after pollination, AGPs are secreted from the pollen cytoplasm into the pollen wall and contact the extracellular matrix of stigmatic tip cells followed by micropylar canal cells; (3) AGPs synthesized in nucellus cells before pollen grain germination are secreted during pollen tube growth into the extracellular matrix, where they can directly interact with male gametophytes; (4) in vitro cultured pollen tube AGPs labeled with LM2 Abs participate in the germination of pollen grain, while AGPs recognized by JIM8 Abs are essential for pollen tube tip growth.

The role of ethylene and transcriptome alterations in response to hypoxia stress in plants / Rola etylenu i zmiany na poziomie transkryptomu w odpowiedzi na stres niedotlenienia u roslin.

Hypoxia in plants is a usually the result of heavy rainfall and the subsequent flooding. All current climate models indicate a notable increase in extreme weather over the coming years. Depending on the species and geographical location, plants have developed two distinct strategies for hypoxia stress adaptation: escape and quiescence. The escape strategy involves rapid growth of part of the shoot above the water level whe­reas the second strategy requires a significant reduction in the metabolic rate of the plant in order to survive until the negative environmental conditions pass. These processes are primarily regulated by ethylen in addition to the transcription factor, ERF (ethylen response factor), which enables the transcription of hypoxia response genes. These processes are primarily regulated by ethylen in addition to the transcription factors, ERFs (ethylen response factors), which enables the transcription of hypoxia response genes. Most ERF genes are constitutively trans­cribed independently of oxygen concentration. However, post-translational modification of the N-terminus of ERFs leads to their degradation in plants growing under physiological conditions. During hypoxia there is an increase in the expression level of genes associated with carbon, nitrogen, glycolysis or anaerobic respiration. However, as shown by studies using ribosome profiling, in order to save energy, plants under hypoxic stress strongly inhibit the process of initiating translation. The regulation of gene expression under stress conditions is also influen­ced by the accumulation of poly(A) RNA in the cell nucleus and cytoplasmic stress granules.

Dynamic distribution of ARGONAUTE1 (AGO1) and ARGONAUTE4 (AGO4) in Hyacinthus orientalis L. pollen grains and pollen tubes growing in vitro.

The transcriptional and posttranscriptional AGO-mediated control of gene expression may play important roles during male monocot gametophyte development. In this report, we demonstrated dynamic changes in the spatiotemporal distribution of AGO1 and AGO4, which are key proteins of the RNA-induced silencing complex (RISC) in Hyacinthus orientalis male gametophyte development. During maturation of the bicellular pollen grains and in vitro pollen tube growth, the pattern of AGO1 localization was correlated with previously observed transcriptional activity of the cells. During the period of high transcriptional activity, AGO1 is associated with chromatin while the clustered distribution of AGO1 in the interchromatin areas is accompanied by condensation of chromatin and the gradual transcriptional silencing of both cells in mature, dehydrated pollen. During pollen tube growth and the restarting of RNA synthesis in the vegetative nucleus, AGO1 is dispersed in the chromatin. Additionally, the gradual increase in the cytoplasmic pool of AGO1 in the elongating pollen tube indicates the activation of the posttranscriptional gene silencing (PTGS) pathway. During pollen tube growth in the generative cell and in the sperm cells, AGO1 is present mainly in the areas between highly condensed chromatin clusters. Changes in the distribution of AGO4 that indicated the possibility of spatiotemporal organization in the RNA-directed DNA methylation (RdDM) process (cytoplasmic and nuclear steps) were also observed during hyacinth male gametophyte development. Based on our findings, we propose that in the germinating pollen tube, the cytoplasmic assembly of AGO4/siRNA takes place and that the mature complexes could be transported to the nucleus to carry out their function during the next steps of pollen tube growth.

Salt stress vs. salt shock - the case of sugar beet and its halophytic ancestor.

BACKGROUND: Sugar beet is a highly salt-tolerant crop. However, its ability to withstand high salinity is reduced compared to sea beet, a wild ancestor of all beet crops. The aim of this study was to investigate transcriptional patterns associated with physiological, cytological and biochemical mechanisms involved in salt response in these closely related subspecies. Salt acclimation strategies were assessed in plants subjected to either gradually increasing salt levels (salt-stress) or in excised leaves, exposed instantly to salinity (salt-shock). RESULT: The majority of DEGs was down-regulated under stress, which may lead to certain aspects of metabolism being reduced in this treatment, as exemplified by lowered transpiration and photosynthesis. This effect was more pronounced in sugar beet. Additionally, sugar beet, but not sea beet, growth was restricted. Silencing of genes encoding numerous transcription factors and signaling proteins was observed, concomitantly with the up-regulation of lipid transfer protein-encoding genes and those coding for NRTs. Bark storage protein genes were up-regulated in sugar beet to the level observed in unstressed sea beet. Osmotic adjustment, manifested by increased water and proline content, occurred in salt-shocked leaves of both genotypes, due to the concerted activation of genes encoding aquaporins, ion channels and osmoprotectants synthesizing enzymes. bHLH137 was the only TF-encoding gene induced by salt in a dose-dependent manner irrespective of the mode of salt treatment. Moreover, the incidence of bHLH-binding motives in promoter regions of salinity-regulated genes was significantly greater than in non-regulated ones. CONCLUSIONS: Maintaining homeostasis under salt stress requires deeper transcriptomic changes in the sugar beet than in the sea beet. In both genotypes salt shock elicits greater transcriptomic changes than stress and it results in greater number of up-regulated genes compared to the latter. NRTs and bark storage protein may play a yet undefined role in salt stress-acclimation in beet. bHLH is a putative regulator of salt response in beet leaves and a promising candidate for further studies.

Salt Stress Reveals a New Role for ARGONAUTE1 in miRNA Biogenesis at the Transcriptional and Posttranscriptional Levels.

Plants as sessile organisms have developed prompt response mechanisms to react to rapid environmental changes. In addition to the transcriptional regulation of gene expression, microRNAs (miRNAs) are key posttranscriptional regulators of the plant stress response. We show here that the expression levels of many miRNAs were regulated under salt stress conditions. This regulation occurred at the transcriptional and posttranscriptional levels. During salinity stress, the levels of miRNA161 and miRNA173 increased, while the expression of pri-miRNA161 and pri-miRNA173 was down-regulated. Under salt stress conditions, miRNA161 and miRNA173 were stabilized in the cytoplasm, and the expressions of MIR161 and MIR173 were negatively regulated in the nucleus. ARGONAUTE1 (AGO1) participated in both processes. We demonstrated that AGO1 cotranscriptionally controlled the expression of MIR161 and MIR173 in the nucleus. Our results suggests that AGO1 interacts with chromatin at MIR161 and MIR173 loci and causes the disassembly of the transcriptional complex, releasing short and unpolyadenylated transcripts.

Regulation of poly(A) RNA retention in the nucleus as a survival strategy of plants during hypoxia.

Last finding indicates that post-transcriptional processes are significant in low-oxygen conditions, but their nature is poorly understood. Here, we localized poly(A) RNA and mRNA coding proteins involved and not involved with resistance to hypoxia in Lupinus luteus and Arabidopsis thaliana during submergence and after recovery of aerobic conditions. We showed a strong nuclear accumulation of poly(A) RNA and 6 of 7 studied mRNAs with a concurrent strong reduction in RNA polymerase II transcription during hypoxia. In this study, the nucleus did not accumulate mRNA of the ADH1 (alcohol dehydrogenase 1) gene, which is a core hypoxia gene. The RNA accumulation in the nucleus is among the mechanisms of post-transcriptional gene regulation that prevents translation. However re-aeration was accompanied by a strong increase in the amount of the mRNAs in the cytoplasm and a simultaneous decrease in nuclear mRNAs. This finding indicates that the nucleus is a storage site for those of mRNAs which are not involved in the response to hypoxia for use by the plants after the hypoxic stress. In this study, the highest intensity of RNA accumulation occurred in Cajal bodies (CBs); the intensity of accumulation was inversely correlated with transcription. Under hypoxia, ncb-1 mutants of Arabidopsis thaliana with a complete absence of CBs died sooner than wild type (WT), accompanied by a strong reduction in the level of poly(A) RNA in the nucleus. These results suggest that the CBs not only participate in the storage of the nuclear RNA, but they also could take part in its stabilization under low-oxygen conditions.

Transcriptional activity in diplotene larch microsporocytes, with emphasis on the diffuse stage.

Manuscript provides insights into the biology of long-lived plants, different from Arabidopsis, tomato or grass species that are widely studied. In the European larch the diplotene stage lasts approximately 5 months and it is possible to divide it into several substages and to observe each of them in details. The diplotene stage is a period of intensive microsporocyte growth associated with the synthesis and accumulation of different RNA and proteins. Larch microsporocytes display changes in chromatin morphology during this stage, alternating between 4 short stages of chromatin condensation (contraction) and 5 longer diffusion (relaxation) stages. The occurrence of a diplotene diffusion stage has been observed in many plant species. Interestingly, they have also been observed during spermiogenesis and oogenesis in animals. The aim of this study was to examine whether chromatin relaxation during the diplotene is accompanied by the synthesis and maturation of mRNA. The results reveal a correlation between the diffusion and chromatin decondensation, transcriptional activity. We also found decreasing amount of poly(A) mRNA synthesis in the consecutive diffusion stages. During the early diffusion stages, mRNA is intensively synthesized. In the nuclei large amounts of RNA polymerase II, and high levels of snRNPs were observed. In the late diffusion stages, the synthesized mRNA is not directly subjected to translation but it is stored in the nucleus, and later transported to the cytoplasm and translated. In the last diffusion stage, the level of poly(A) RNA is low, but that of splicing factors is still high. It appears that the mRNA synthesized in early stages is used during the diplotene stage and is not transmitted to dyad and tetrads. In contrast, splicing factors accumulate and are most likely transmitted to the dyad and tetrads, where they are used after the resumption of intense transcription. Similar meiotic process were observed during oogenesis in animals. This indicates the existence of an evolutionarily conserved mechanism of chromatin-based regulation of gene expression during meiotic prophase I.

Dedifferentiation of Arabidopsis thaliana cells is accompanied by a strong decrease in RNA polymerase II transcription activity and poly(A+) RNA and 25S rRNA eradication from the cytoplasm.

The mechanisms of plant cell dedifferentiation and the acquisition of totipotency are poorly understood. One of the methods to induce the dedifferentiation process in plant cells is simple and requires the removal of the cell wall. After cell wall removal in protoplasts, large-scale chromatin decondensation is observed (Tessadori et al. in J Cell Sci 120:1200-1208, 2007). Here, we show that in Arabidopsis thaliana protoplasts, despite chromatin decondensation, RNA polymerase II transcriptional activity is reduced. The subsequent investigated stages displayed a clear decrease in the quantity of 25S ribosomal RNA (rRNA) first and then poly(A+) RNA, particularly in the cytoplasm. Therefore, the reduced transcription activity and the removal of these RNA transcripts from the cytoplasm is a crucial process in obtaining totipotency in plant cells. After the cytoplasm cleaning of transcripts derived from mesophyll cells, we observed the resynthesis of these RNAs. An increase in the amount of examined molecules to a level similar to that in differentiated mesophyll cells precedes the divisions of already undifferentiated cells. In this work, we show changes in RNA polymerase II transcription dynamics and the quantity of poly(A+) RNA and 25S rRNA during dedifferentiation and re-entry into the cell cycle.

Poly(A) RNAs including coding proteins RNAs occur in plant Cajal bodies.

The localisation of poly(A) RNA in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus, Arabidopsis thaliana) nuclei was studied through in situ hybridisation. In both types of nuclei, the amount of poly(A) RNA was much greater in the nucleus than in the cytoplasm. In the nuclei, poly(A) RNA was present in structures resembling nuclear bodies. The molecular composition as well as the characteristic ultrastructure of the bodies containing poly(A) RNA demonstrated that they were Cajal bodies. We showed that some poly(A) RNAs in Cajal bodies code for proteins. However, examination of the localisation of active RNA polymerase II and in situ run-on transcription assays both demonstrated that CBs are not sites of transcription and that BrU-containing RNA accumulates in these structures long after synthesis. In addition, it was demonstrated that accumulation of poly(A) RNA occurs in the nuclei and CBs of hypoxia-treated cells. Our findings indicated that CBs may be involved in the later stages of poly(A) RNA metabolism, playing a role storage or retention.

The perichromatin region of the plant cell nucleus is the area with the strongest co-localisation of snRNA and SR proteins.

The spatial organisation of the splicing system in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus) nuclei was studied by immunolabelling of SR proteins, snRNA, and the PANA antigen, known markers for interchromatin granule clusters in mammalian cells. Electron microscope results allowed us to determine the distribution of these molecules within the structural domains of the nucleus. Similar to animal cells, in both plant species SR proteins were localised in interchromatin granules, but contrary to animal cells contained very small amounts of snRNA. The area with the strongest snRNA and SR protein co-localisation was the perichromatin region, which may be the location of pre-mRNA splicing in the plant cell nuclei. The only observable differences in the organisation of reticular and chromocentric nuclei were the size of the speckles and the number of snRNA pools in the condensed chromatin. We conclude that, despite remarkable changes in the nuclear architecture, the organisation of the splicing system is remarkably similar in both types of plant cell nuclei.

Ultrastructural differences in rectus sheath of hernia patients and healthy controls.

BACKGROUND: The etiology of inguinal hernia remains unclear. Research data indicate the presence of pathologic alterations within the connective tissue; their exact character remains the subject of dispute. The search for new methods to diagnose connective tissue abnormalities, and thoroughly explain the character of the ultrastructural alterations, continues. MATERIALS AND METHODS: The study group included 10 male patients aged 18-60 y (five with primary inguinal hernia and five with acute appendicitis with no history of hernia). A specimen of the rectus muscle sheath was harvested from all of them upon surgery. The tissue samples were fixed and examined by spectrofluorometry and fluorescence microscopy, yielding fluorescence spectra and microscopic fluorescence images. RESULTS: Both techniques have demonstrated significant differences between the biopsy samples harvested from hernia patients and healthy controls. The groups of fluorescence spectra were shifted relative to each other and showed maximum emission at different wavelengths after excitation with 350 nm light (arbitrarily chosen for one of the cross-link proteins). The spectra obtained for healthy controls were more homogenous, while the spectra of the hernia samples differed even between each other. In microscopic images, the difference was a more chaotic distribution of fluorophores in the samples obtained from hernia patients. CONCLUSIONS: The evidence of significant differences between the samples harvested from the same location from hernia patients and healthy controls, found by fluorescence techniques, indicates the presence of abnormalities in the connective tissue forming the rectus muscle sheath. This area is not a part of the hernial defect, therefore, we can assume that the changes can be attributed to a generalized process.

Transcribed DNA is preferentially located in the perichromatin region of mammalian cell nuclei.

The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.

Calreticulin expression and localization in plant cells during pollen-pistil interactions.

In this report, the distributions of calreticulin (CRT) and its transcripts in Haemanthus pollen, pollen tubes, and somatic cells of the hollow pistil were studied. Immunoblot analysis of protein extracts from mature anthers, dry and germinated pollen, growing pollen tubes, and unpollinated/pollinated pistils revealed a strong expression of CRT. Both in vitro and in situ studies confirmed the presence of CRT mRNA and protein in pollen/pollen tubes and somatic cells of the pistil transmitting tract. The co-localization of these molecules in ER of these cells suggests that the rough ER is a site of CRT translation. In the pistil, accumulation of the protein in pollen tubes, transmitting tract epidermis (tte), and micropylar cells of the ovule (mc) was correlated with the increased level of exchangeable calcium. Therefore, CRT as a Ca(2+)-binding/buffering protein, may be involved in mechanism of regulation calcium homeostasis in these cells. The functional role of the protein in pollen-pistil interactions, apart from its postulated function in cellular Ca(2+) homeostasis, is discussed.

Nuclear bodies in Douglas fir (Pseudotsuga menziesii Mirb.) microspores.

The identification of nucleolar proteins and immunocytochemical localization of small nuclear ribonucleoprotein (snRNP) elements revealed the presence of three types of nuclear bodies in Douglas fir microspore nuclei. One type consists of structures resembling Cajal bodies (CBs) and contains nucleolar proteins as well as snRNPs and U2 snRNA. The second type is bizonal bodies, which are nuclear bodies also linked with the splicing system. The bizonal body comprises two parts: the first contains Sm proteins and stains strongly with silver stain, and the second resembles CBs in terms of the degree of silver staining and molecular composition. Douglas fir is the second species after larch where the presence of bizonal bodies has been demonstrated. Pseudotsuga menziesii Mirb and Larix decidua Mill are species with one of the longest microsporogenesis processes known in plants. The presence of bizonal bodies in both species may be linked to the intensification of the splicing processes in microspores with an exceptionally long cell cycle. The third type of structure is dense bodies, whose morphology and degree of silver staining strongly indicate their functional and spatial relationship to the dense part of bizonal bodies.

New type of snRNP containing nuclear bodies in plant cells.

In larch (Larix decidua Mill.) microspores a new type of nuclear bodies has been found which are an element of the spatial organization of the splicing system in plant cell. These are bizonal bodies, ultrastructurally differentiated into a coiled part and a dense part. Using immunocytochemistry and in situ hybridization at the EM level, the coiled part of the bizonal body was found to contain snRNA including U2 snRNA, Sm proteins and nucleolar proteins of the agyrophilic type and fibrillarin. The dense part contains Sm proteins but lacks snRNA. Such a separation of macromolecules related to splicing occurring within the bizonal bodies microspore is striking by the similarity of these bodies to amphibian oocyte snurposomes. The occurrence in plant cells, beside widely known coiled bodies (CBs), also of other nuclear bodies related to splicing proves that in plants similarly as for animals the differentiation among domains containing elements of the splicing system occurs.